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A fungal protease and use thereof

A technology of serine protease and fungus, applied in hydrolytic enzymes, biochemical treatment of enzymes/microorganisms, introduction of foreign genetic material using vectors, etc., can solve problems that have not been studied

Active Publication Date: 2012-08-01
AB ENZYMES GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Their use in other biotechnological processes remains unstudied

Method used

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  • A fungal protease and use thereof
  • A fungal protease and use thereof
  • A fungal protease and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0209] Embodiment 1. Trichoderma reesei QM6a prb1 ( Tr prb1 ) and Fusarium graminearum ALKO1726 FgprtS8A Cloning of the protease gene

[0210] (a) DNA isolation and molecular biology methods used

[0211] Standard molecular biology methods are used for isolation and enzymatic treatment of DNA (eg, isolation of plasmid DNA, digestion of DNA to generate DNA fragments), transformation in E. coli, sequencing, etc. Basic methods used are as described by the enzyme, reagent or reagent expression cassette manufacturer, or as described in standard molecular biology handbooks such as Sambrook and Russell (2001). Isolation of genomic DNA from T. reesei QM6a and F. graminearum ALKO1726 was performed as described by Raeder and Broda (1985). The phenol extraction phase was performed using PhaseLock tubes (Eppendorf, Germany).

[0212] (b) Oligonucleotide primers for gene cloning

[0213] Cloning by PCR using Trichoderma reesei QM6a and Fusarium graminearum ALKO1726 genomic DNA pre...

Embodiment 2

[0223] Example 2. Production of Tr Prb1 and Fg_ALKO1726 recombinant protease in Trichoderma reesei

[0224] (a) Preparation of expression cassette and its transformation into Trichoderma reesei

[0225] by separately amdS ( Acetamidase) marker gene was connected to plasmid pALK2650 and pALK2707 (Example 1c) to construct the expression plasmid pALK2701 ( Tr prb1 ) and pALK2708 ( FgprtS8A ). amdS marked in cbh1 The terminator is ligated into the expression construct. Similar constructions have been described eg in Paloheimo et al. (2003). In expression cassettes pALK2701 and pALK2708 ( image 3 with 4 ), by PCR will have its own signal sequence Tr prb1 with FgprtS8A Trichoderma reesei cbh1 (cel7A) Promoter exact fusion. Use the one generated after the stop codon in PCR Bam HI site, connecting the 3-' end of the gene with cbh1 Terminator fusion. This is in the build in cbh1 The original termination sequence is not left before the terminator sequence. us...

Embodiment 3

[0231] Example 3. Purification and characterization of recombinant Tr Prb1 and Fg_ALKO1726 proteases

[0232] Cells and solids were removed from the spent medium obtained from the fermentation (Example 2) by centrifugation for 30 min at 4°C, 50000 g (Sorvall RC6 plus). 15 ml of supernatant was used for protease purification. All purification steps were performed in a cold room. After centrifugation, samples were filtered through a 0.44 μm filter (MILLEX HV Millipore) before being applied to a HiPrep 26 / 10 Desalting column (from GE Healthcare) equilibrated in 20 mM Tris pH 8.8. Gel filtered samples were applied to a 20 mL Q Sepharose FF column (from GE Healthcare) equilibrated in 20 mM Tris pH 8.8. The effluent fraction with proteolytic activity was concentrated using an Amicon Ultra centrifugal filter unit 10000 MWCO (Millipore). The concentrated sample was applied to a Superdex 75 10 / 300 GL column (GE Healthcare) and eluted with 20 mM Hepes, 150 mM NaCl pH 6,8. Protease-c...

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Abstract

The present invention is related to a fungal serine protease enzyme useful in modification, degradation or removal of proteinaceous material, which enzyme comprises an amino acid sequence of the mature Tr Prb 1 enzyme having an amino acid sequence of SEQ ID NO: 10 or a variant thereof having similar activity. The serine protease is obtainable from Trichoderma. Also disclosed are nucleic acid sequences encoding said protease, such as plasmid pALK2650 comprising the nucleotide sequence SEQ ID NO: 10 of the full length enzyme deposited in E. coli RF8052 under accession number DSM 22635. Said protease is useful as an enzyme preparation applicable in detergent compositions and for treating fibers, for treating wool, for treating hair, for treating leather, for treating food or feed, or for any applications involving modification, degradation or removal of proteinaceous material at low or moderate temperature ranges.

Description

field of invention [0001] The present invention relates to fungal serine proteases useful in a variety of industrial applications, particularly in laundry and dishwashing detergents, wherein the performance of the enzymes at low or moderate temperature ranges is advantageous. The present invention relates to isolated nucleic acid molecules encoding the enzymes, recombinant vectors, host cells for producing the enzymes, enzyme compositions comprising the enzymes and methods for preparing such compositions. The invention also relates to various uses of said enzymes or compositions comprising said enzymes. Background technique [0002] Microbial extracellular proteases account for the majority, more than one-third of total industrial enzyme sales worldwide (Cherry and Fidantsef, 2003). About 90% of commercial proteases are detergent enzymes (Gupta et al., 2002). Other applications include eg food, feed, leather, pharmaceuticals, diagnostics, waste management and silver recove...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/11C12P21/02C12R1/00C12R1/885A23L29/00
CPCC11D3/386C12N9/58C11D3/38681A23L29/06C12N15/52C12N15/80D06M16/003
Inventor L.瓦尔塔卡里K.容图南S.马基南P.奥贾帕洛M.帕罗海莫
Owner AB ENZYMES GMBH
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